The major objective of this proposal is to examine the mechanisms by which cocaine and its major metabolites may injure myocardial cells. We plan to investigate cocaine-induced myocardial cell injury with in vitro systems of postnatal and adult rat myocardial cells in primary culture. Two lethal cardiovascular responses to cocaine include myocardial infarction and ventricular fibrillation. The mechanisms thought to be responsible for these cardiotoxic effects of cocaine have been correlated to its sympathomimetic effects (inhibition of neuronal uptake of norepinephrine) and local anesthetic properties (Na+ channel blockade). In contrast, our laboratory has suggested that cocaine and/or its metabolites may have direct cardiotoxic effects which are unrelated to those mechanisms described above. Whereas cocaine's induction of myocardial infarction or cellular necrosis is thought to be related to sympathomimetic vascular constriction and ensuing ischemic episodes, our laboratory has demonstrated that cocaine may be directly toxic to myocytes lacking vascular components and sympathetic innervation. This proposal will focus on myocardial cell injury produced by cocaine and its major metabolites and the mechanistic role that alterations in cytosolic and mitochondrial calcium homeostasis may have in affecting myocardial cell integrity, especially the function and activity of mitochondria. We hypothesize that cocaine may disrupt mitochondrial regulation of calcium and lead to the formation of dysfunctional mitochondria which show a loss of mitochondrial membrane potential and a reduction in ATP formation. After exposure of the myocardial cell cultures to different concentrations and exposure periods of cocaine and its major metabolites, the integrity of the cells will be assessed by the following methods: cell viability and plasma membrane integrity (leakage of cytosolic enzymes, neutral red uptake, and the reduction of 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl tetrazolium bromide, MTT); contractile activity and cell morphology as evaluated by phase contrast microscopy; cytosolic and mitochondrial calcium alterations as evaluated by Fura-2 and digitized fluorescence imaging (DFI); cellular metabolism and energy status (cytosolic lactate/pyruvate ratios and cellular ATP/ADP ratios); and mitochondrial function as evaluated by respiratory control ratios (state 3 respiration/state 4 respiration), normal production of superoxide, and the determination of mitochondrial inner membrane potential with rhodamine 123 and DFI. By correlating changes in cell viability, cellular metabolism, and calcium levels with discrete events in mitochondrial function and activity over specific time periods of exposure to cocaine and its major metabolites, we expect to better elucidate the mechanism(s) of cocaine-induced myocardial toxicity under carefully controlled conditions.